Lipophilic oligosaccharide antibiotic salt compositions

ABSTRACT

Pharmaceutically acceptable compositions of matter comprising a lipophilic oligosaccharide antibiotic, e.g., the everninomicin-type antibiotic of Formula III, at least a stoichiometric amount of a base, e.g., N-methylglucamine, an amount of, e.g., hydroxypropyl-β-cyclodextrin, and optionally a pharmaceutically acceptable non-ionic surfactant, e.g., Tween-80, pharmaceutical compositions containing such compositions of matter, methods of treating and preventing susceptible bacterial infections in animals especially human beings as well as a method of preventing adverse reaction syndrome while simultaneously delivering an antiinfective amount of a lipophilic oligosaccharide antibiotic such as that of Formula III to said animals as well as the use of the compositions of matter for the preparation of a medicament for such treating or preventing are disclosed. ##STR1##

The present application is the U.S. national application correspondingto International Application No, PCT/US 92/08565, filed Oct. 14, 1992and designating the U.S., which PCT application is in turn acontinuation-in-part of U.S. application Ser. No. 07/777,864, filed Oct.16, 1991, now abandoned.

The present application is the U.S. national application correspondingto International Application No, PCT/US 92/08565, filed Oct. 14, 1992and designating the U.S., which PCT application is in turn acontinuation-in-part of U.S. application Ser. No. 07/777,864, filed Oct.16, 1991, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel compositions of matter comprising alipophilic oligosaccharide antibiotic, and to pharmaceuticalformulations containing such compositions of matter and to methods ofmaking and using such pharmaceutical compositions to treat and/orprevent microbial infections in animals especially mammals such as humanbeings.

Lipophilic oligosaccharide antibiotics including, for example,everninomicins, curamycins, avilamycins and flambamycins are members ofthe orthosomycin family of antibiotics which contain at least one acidicphenolic hydrogen, and two orthester linkages associated withcarbohydrate residues. See for example. A. K. Ganguly in "Kirk-Othmer,Encyclopedia of Chemical Technology", (1978), Volume 2, pp. 205-209,Third Edition, John Wiley and Sons and W. D. Oillis, et al., Tetrahedron(1979), Volume 35, pp. 105-127. These lipophilic oligosaccharideantibiotics exhibit broad spectrum biological activity against grampositive and some gram negative bacteria in various in vitro-assays, andin-vivo activity in animal models such as mice, but to date nopharmaceutically acceptable formulation of such antibiotics useful forvivo administration has been available. Thus, we have observed thatinjection of these lipophilic oligosaccharide antibiotics cause anadverse reaction syndrome. The term "adverse reaction syndrome" as usedherein means symptoms of the following type observed in animals such asmice upon parenteral administration of lipophilic oligosaccharideantibiotics: incoordination, ataxia, lateral recumbency, urination, hindleg rigidity, labored breathing, and arrest. Thus, in summary, there areno known pharmaceutically acceptable compositions of these lipophilicoligosaccharide antibiotics for the safe and effective use of thesepotent antibiotics in animals including mammals such as human beings.

Cyclodextrins are modified starches made from glucopyranose units andinclude α-cyclodextrin consisting of six glucopyranose units,β-cyclodextrin consisting of seven glucopyranose units, andγ-cyclodextrin consisting of eight glucopyranose units. The α-, β- andγ-cyclodextrins and derivatives thereof have an inside surface or cavitywhich is lipophilic and an outer surface which is hydrophilic. Thiscombination of a hydrophobic cavity and a hydrophilic outer surface hasled to the use of cyclodextrins and derivatives thereof for themolecular complexation or encapsulation of many hydrophobic and/orunstable drugs of suitable dimensions, thereby improving solubility,stability and bioavailability of such drugs. Derivatives of α-, β- andγ-cyclodextrins for example hydroxypropyl-β-cyclodextdrins are disclosedby Jozsef Szejtli in Pharmaceutical Technology, Jun. 1991, 36-40.

Complexes of α-, β- and γ-cyclodextrins, mixtures and derivativesthereof are disclosed in, for example, N. Bodor U.S. Pat. No. 4,983,586.The Bodor U.S. Pat. No. 4,983,586 discloses a method of decreasing theincidence of precipitation of a lipophilic or water labile drugoccurring at/or near the injection site and/or in the lungs followingparenteral administration, by parenterally administering said drug in anaqueous solution containing a large quantity, i.e., 20 to about 50weight percent of hydroxypropyl-β-cyclodextrin.

Josef Pitha in U.S. Pat. No. 4,727,064 and The International J. ofPharmaceutics, (1986) 29, 73-82 disclose the use of a concentrated,i.e., 40-60 weight percent, aqueous solution ofhydroxypropyl-β-cyclodextrin to solubilize various drugs such asacetaminophen, sex steroids, cardiac glycosides such as digoxin, as wellas retinoic acid and acid salts thereof. See also the Pitha U.S. Pat.No. 4,596,795 which discloses the administration of the sex hormones,testosterone, progesterone and estradiol as complexes withpoly-β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

The Bodor and Pitha references make no reference to phenols orlipophilic oligosaccharide antibiotics.

Janssen Pharmaceutica N. V. International Pat.ent Application No.PCT/EP84/00417 published under International Publication No. WO 85/02767on 4, Jul. 1985 discloses pharmaceutical compositions comprisingcomplexes of drugs which are unstable or only sparingly soluble in waterwith partially etherified β-cyclodextrin ("β-CD") of the Formula(β-CD)-OR wherein the residue R is hydroxyethyl, hydroxypropyl,dihydroxypropyl and part of the residue R may optionally be alkylgroups, especially methyl or ethyl.

If the drug molecule has basic or add groups which may possibly be usedto increase water solubility by salt formation, the JanssenInternational Publication No. WO 85/02767 teaches that salt formation asa rule results in decreased efficacy or impaired chemical stability andthus, salt formation to solubilize poorly water soluble acidic and basiccompounds is discouraged. There is no disclosure of lipophilicoligosaccharide antibiotics or the compositions of the presentinvention.

Various strains of bacteria such as gram-positive cocci, e.g.,streptococci and enterococci as well as methicillin-resistant andmethicillinsusceptible staphylococci have become resistant tocommercially available antibiotics, e.g., vancomycin.

Thus, there is a need for pharmaceutically acceptable compositions fortreating bacterial infections including methicillin-resistant andmethicillin-susceptible stapliylococci and vancomycin-resistantbacteria. There is also a need for pharmaceutically acceptablecompositions containing a lipophilic oligosaccharide antibiotic activeagainst a broad range of susceptible gram-positive and gram-negativebacterial infections, especially pharmaceutical compositions adapted forparenteral use which avoid occurrence of the adverse reaction syndrome.

BRIEF SUMMARY OF THE INVENTION

Surprisingly, we have discovered a means by which lipophilicoligosaccharide antibiotics having good antibacterial activity againstsusceptible gram-positive and/or gram-negative bacterial infections maybe delivered to animals, especially mammals such as man afflicted withsusceptible gram-positive or gram-negative bacterial infections toprovide effective treatment and/or prevention thereof whilesimultaneously avoiding occurrence of the adverse reaction syndrome.This means comprises combining a lipophilic oligosaccharide antibioticwith at least about a stoichiometric amount of a specified base and anamount of an agent such as hydroxypropyl-α-, -β- or -γ-cyclodextrinhaving about 2 to 15 hydroxypropyl groups per molecule of cyclodextrinsufficient to achieve efficacious delivery of the lipophilicoligosaccharide antibiotic to the serum of an animal whilesimultaneously avoiding adverse reaction syndrome.

SUMMARY OF THE INVENTION

The present invention provides a composition of matter comprising:

(a) a lipophilic oligosaccharide antibiotic represented by Formula I;##STR2## wherein ##STR3## X is one of NO₂, NO, NH₂, NHCOCH₃, NHOH, NH(C₂H₅), N(C₂ H₅)₂, OH or H;

R₂ is one of ═CH₃, COCH(CH₃)₂, COCH₃, CO(CH₂)₃ CH₃, COCH₂ CH₃ or H;

R₃ is CH₃ or H;

R₄ is one of COCH₃, CH(OCH₃)(CH₃), CH(OH)CH₃, CHO, or H; ##STR4## R₆ isCH₃ or H; R₇ is CH₃ or H;

R₈ is CH₃, CH₂ OH or H

R₉ is CH₃ or H;

Y is OH, CH₃, or H;

W is C1 or H; and

Z is C1 or H.

(b) at least about a stoichiometric amount of a base capable of forminga pharmaceutically acceptable salt with a lipophilic oligosaccharideantibiotic of Formula I;

(c) an amount of dimethylsulfoxide, glycerol, a sorbitanmono-9-octadecenoate poly(oxy-1,2-ethanediyl)derivative, dextran, ahydroxypropyl-α-, -β- or -γ-cyclodextrin wherein the average number ofhydroxypropyl substituents on said and α-, β- and γ-cyclodextrin is inthe range of about 2 to about 15, and said amount is sufficient toachieve efficacious delivery of said lipophilic oligosaccharideantibiotic to the serum of an animal while simultaneously avoidingadverse reaction syndrome; and

(d) 0 to 6.0% by weight (basis, said antibiotic of Formula I) of apharmaceutically acceptable non-ionic surfactant.

The present invention more preferably provides a composition of mattercomprising

(a) a compound represented by the Formula II ##STR5## wherein X is oneof NO₂, NO, NHOH, NH₂, NHCOCH₃, NHC₂ H₅, N(C₂ H₅)₂, OH or H

Y is OH, CH₃ or H

R₂ is H or CH₃

R₃ is H

R₄ is H or CH(OCH₃)(CH₃)

and ##STR6## (b) at least about a stoichiometric amount of a basecapable of forming a pharmaceutically acceptable salt with a lipophilicoligosaccharide antibiotic of Formula II;

(c) an amount of hydroxypropyl-α-, -β- or -γ-cyclodextrin wherein theaverage number of hydroxypropyl substituents on said α-, -β- andγ-cyclodextrin is in the range of about 2 to about 15, and said amountis sufficient to achieve efficacious delivery of said lipophilicoligosaccharide antibiotic to the serum of an animal whilesimultaneously avoiding occurrence of adverse reaction syndrome; and

(d) 0 to 6.0% by weight (basis, said antibiotic of Formula II) of apharmaceutically acceptable non-ionic surfactant.

The present invention further provides a composition of mattercomprising

(a) the antibiotic compound represented by Formula III ##STR7## (b) atleast about two equivalents of a base (per mole of the compound ofFormula III) capable of forming a pharmaceutically acceptable salt ofthe compound of Formula III) (c) an amount of hydroxypropyl-α-, -β- or-γ-cyclodextrin having about 2 to about 15 hydroxypropyl groups permolecule of said -α-, -β- or -γ-cyclodextrin and wherein said amount ofsaid cyclodextrin is sufficient to achieve efficacious delivery of saidlipophilic oligosaccharide antibiotic to the serum of an animal whilesimultaneously avoiding occurrence of adverse reaction syndrome; and (d)0 to 6.0% by weight (basis, said antibiotic of Formula III) of apharmaceutically acceptable non-ionic surfactant.

Pharmaceutical compositions formed by admixing a composition of mattercomprising a compound represented by Formulas I, II or III and at leastabout a stoichiometric amount of a base capable of forming apharmaceutically acceptable salt thereof and an amount of ahydroxypropyl-α-, -β- or -γ-cyclodextrin with a pharmaceuticallyacceptable carrier as well as methods of using such pharmaceuticalcompositions for treating or preventing susceptible gram positive andgram negative bacterial infections in animals, especially mammals inneed of such treating or preventing are also provided. "The preferredpharmaceutical composition of this invention contains the following: (a)the lipophilic oligosaccharide antibiotic represented by Formula III,(b) a base capable of forming a pharmaceutically acceptable salt withlypophilic oligosacharide antibiotic of Formula III and (c)hydoxypropyl-α-, -β- or -γ-cyclodextrin having about 2 to about 15hydroxypropyl groups per molecule of said α-, βand γ-cyclodextrin andwherein the molar ratio of (a):(b):(c) is 1:2-3:1-6."

As a preferred form of the invention, the aforesaid pharmaceuticalcompositions are particularly applicable to parenteral administration,especially in vivo administration to human beings by the intravenous(IV) route.

The present invention also provides a method of preventing adversereaction syndrome in animals following parenteral injection of alipophilic oligosaccharide antibiotic represented by Formula I, II orIII while simultaneously delivering an antiinfective amount of saidantibiotic to an animal, which method comprises parenterallyadministering to said animal an amount of a composition of matter ofthis invention sufficient for such purpose together with apharmaceutically acceptable carrier.

The present invention further provides the use of a lipophilicoligosaccharide antibiotic represented by Formula I for the preparationof a medicament for treating or preventing susceptible gram positive andgram negative bacterial infections in animals, especially mammalsin-need of such treating or preventing.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically illustrates the progression, with time, of a typicalfermentation of Micromonospora carbonacea. var. africana, NRRL 15099,ATCC 39149.

DETAILED DESCRIPTION OF THE INVENTION AND OF THE PREFERRED EMBODIMENTS

Lipophilic oligosaccharide antibiotics, for example everninomicinantibiotics, exhibit useful in vitro antibacterial activity but do notreadily form complete aqueous solutions suitable for safe and effectivein vivo administration (i.e. without occurrence of adverse reactionsyndrome). Moreover, salts of these antibiotics formed by admixing atleast about a stoichiometric amount of a base useful in this invention,e.g. the base, N-methylglucamine ("NMG") do not form complete aqueoussolutions, at useful pH values. When said salts were added to water atuseful concentrations of salt, we observed that only colloidaldispersions were formed. These colloidal dispersions tended toaggregate, and eventually gelled, especially in the presence of absorbedcarbon dioxide and when the pH of such colloidal dispersions was lessthan about 9.3. We observed that complete aqueous solutions were formedby increasing the molar ratio of NMG to the compound of Formula III from2:1 or 3:1 up to 12:1, but that the solution so formed with the 12:1molar ratio had an undesirably high pH, was highly buffered and wasirritating. Thus, we observed that parenteral injection into rats orhigher primates, such as monkeys of an aqueous formulation containing 12moles of NMG per one mole of the compound of Formula did not result inadverse reaction syndrome (presumably caused by gelling andprecipitation of the compound of Formula III). The formulation, producedirritation upon injection, which irritation is presumably caused by thelarge amount of NMG base and resulting high pH at the injection site.However, parenteral administration of a composition containing 3 or 5moles of NMG per mole of the compound of Formula III gave rise toadverse reaction syndrome. Whereas various cyclodextrin agents are knownto assist in achieving solutions of specific drugs, (e.g. as theaforementioned Bodor and Pitha references) use of representative agentsof this class by themselves failed to achieve the desired result forlipophilic oligosaccharide antibiotics. Thus, for instance, the use of aspecific cyclodextrin derivative, and a specifichydroxypropyl-β-cyclodextrin per mole of the everninomicin typeantibiotic of Formula III failed to produce a true aqueous solution whenused in the ratio of 6 moles of the specifichydroxypropyl-β-cyclodextrin per mole of the everninomicin-typeantibiotic of formulation III. Surprisingly, we have found that acomposition of matter comprising a lipophilic oligosaccharide antibioticcompound of Formula III, a specified amount of a specific base, e.g.,NMG and a specified agent, e.g., hydroxypropyl-β-cyclodextrin inspecified amounts provides, when admixed with a pharmaceuticallyacceptable carriers, especially water, a formulation which may be usedsafely and effectively for in vivo administration. Surprisingly, we havefound that when 1 mole of a lipophilic oligosaccharide antibiotic, e.g.,the compound of Formula III was admixed with at least 2 to about 12moles of a suitable base, e.g., NMG, in water and with 6 moles of ahydroxypropyl-β-cyclodextrin, e.g. , one having about 3 to 7.5hydroxypropyl groups per molecule of β-cyclodextrin, a clear aqueoussolution of the complex (as measured by light scattering nephelometryand line width measurements in the proton NMR; See Example 4) was formedand the parenteral injection of such complexes into animals did notcause adverse reaction syndrome even at high doses, i.e., 800 mg of suchcomplex per kg of body weight. See Table 4.

As will be evident from the in vivo results summarized in Table 1parenteral injections of the aqueous dispersions of salts, e.g., the NMGsalt of the everninomicin-type antibiotic of Formula III into mice andmonkeys gave rise to the adverse reaction syndrome. Only when theaqueous solutions of one of the compositions of matter of this inventionsuch as one containing a specified complexing agent, e.g.,hydroxypropyl-β-cyclodextrin having about 3 to 7.5 hydroxypropyl groupsper molecule of β-cyclodextrin with the NMG everninomicin-typeantibiotic of Formula III salts were injected into the animals was theoccurrence of adverse reaction syndrome wholly avoided.

Table 2 shows that adverse reaction syndrome can be reduced orcompletely avoided by parenteral injection into mice of clear aqueoussolutions of NMG salts of the everninomicin-type antibiotic of FormulaIII with the specified, e.g., hydroxypropyl-β-cyclodextrin agents ofthis invention.

Table 3 illustrates that increasing the molar ratio of base to theeverninomicin-type antibiotics of Formula III from 2:1 to 9:1 whollyeliminates the occurrence of adverse reaction syndrome at allconcentrations tested upon parenteral injection into mice.

COMPARATIVE EXAMPLE

                  TABLE 1                                                         ______________________________________                                        THE OCCURRENCE OF ADVERSE REACTION                                            SYNDROME AFTER ADMINISTRATION OF AQUEOUS                                      FORMULATIONS OF ONE MOLE OF THE COMPOUND                                      OF FORMULA III: AND OF 2 MOLES NMG WITH AND                                   WITHOUT HP-β-CD.sup.1                                                    Mice             % Adverse Reaction Syndrome.sup.2                            (Concentration of III Injected:                                                                at the following DOSES (MPK.sup.3)                           80 mg/mL)        100.sup.3                                                                              200.sup.3                                                                            500.sup.3                                                                            800.sup.3                             ______________________________________                                        III.sup.4 : 2NMG 100      --     --     --                                    III: 2NMG: 6 HPβCD.sup.5                                                                   0       0      0      0                                     ______________________________________                                        Cynomolgus Monkeys                                                            (Concentration of III Injected:                                               50 mg/mL)        10.sup.6 40.sup.6                                                                             80.sup.6                                     ______________________________________                                        III: 2NMG         50      --     --                                           III: 5NMG        --       33     50                                           III: 2NMG: 6 HPβCD.sup.5                                                                  --       0      0                                            ______________________________________                                         Footnotes to Table 1                                                          .sup.1 HPCD is hydroxypropylcyclodextrin containing 7.4 hydroxypropyl         groups per CD molecule.                                                       .sup.2 Adverse Reaction Syndrome symptoms were observed in the animals        within 2 minutes after IV injection. (single dose)                            .sup.3 MPK is mg of drug per kg of body weight of the mice (groups of 5 t     10, CF1, average weight 20 g, Harlan Sprague  Dawley fasted 18 hours.)        .sup.4 III is the everninomicintype antibiotic compound represented by        Formula III.                                                                  .sup.5 Complex formed. See Examples 3 and 4.                                  .sup.6 MPK is mg of drug per kg of body weight of cynomolgus monkey           (weight range 2.9 to 9.6 kg, Schering Corporation Colony, fasted 18           hours). 3 monkeys were used in the 40 MPK, 5NMG experiment; 2 monkeys wer     used in the other two experiments.                                       

                  TABLE 2                                                         ______________________________________                                        Adverse Reaction Syndrome (ARS).sup.1 Upon Administration of                  Aqueous Formulations of One mole of the Compound of Formula                   III and 2 Moles of NMG and Selected Additives                                 % ARS at the following doses (MPK.sup.2) of Drug of Formula III               (20 mg/ml) In Mice                                                            "Drug of Formula III                                                          Injected"         100      200     250  300                                   ______________________________________                                                  Tween.sup.3                                                                   (Concentration)                                                     III: 2NMG 0           10       100        100                                 III: 2NMG 0.1%        10       100        100                                 III: 2NMG  0.25%      80       100        100                                 III: 2NMG 0.5%        100      100        100                                 III: 2NMG 1%          100      100        100                                 III: 2NMG 2%          --        0     0    40                                 III: 2NMG 3%          0         0     0    0                                            DMSO.sup.4                                                                    (Concentration)                                                     III: 2NMG 0           0         90        100                                 III: 2NMG 1%          0         40   100  100                                 III: 2NMG 5%          0         10   20    50                                 III: 2NMG 10%         0         0    10    50                                           Glycerol                                                                      (Concentration)                                                     III: 2NMG 0           0        100        100                                 III: 2NMG 5%          0         90   90   100                                 III: 2NMG 10%         0         60   100  100                                           Dextran 40.sup.5                                                              (Concentration)                                                     III: 2NMG 0           0        100        --                                  III: 2NMG 1%          20       100        --                                  III: 2NMG 10%         0         50   70   100                                           Dextran 706                                                                   (Concentration)                                                     III: 2NMG 6%          0         90   100  --                                            HPβCD.sup.7                                                              (Molar Ratio)                                                       III: 2NMG 0           0        100        100                                 III: 2NMG 1:0.5                 0          50                                 III: 2NMG 1:1.5                 0          60                                 III: 2NMG 1:2.5                 0          30                                 III: 2NMG 1:3                   0          10                                 III: 2NMG 1:3                              10                                 III: 2NMG 1:6                              0                                  ______________________________________                                         Footnotes to TABLE 2                                                          .sup.1 Adverse Reaction Syndrome Symptoms observed in the mice (groups of     5 to 10, CF1, average weight 20 g, Harlan Sprague Dawley fasted 18 hours)     within 2 minutes after IV injection. (single dose)                            .sup.2 MPK is mg of drug substance of Formula III per kg of animal body       weight.                                                                       .sup.3 Tween is Sorbatan mono9-octadecanoate poly (oxy1,2-ethanediyl) or      as Polysorbate 80 available from ICI Americas Inc., Wilmington Delaware       under the tradename Tween 80.                                                 .sup.4 DMSO is dimethyl sulfoxide                                             .sup.5 Dextran 40 is a high molecular weight (40,000) polymer of glucose      available from Sigma Chemical.                                                .sup.6 Dextran 70 is a high molecular weight (70,000) polymer of glycose      available from Sigma Chemical.                                                .sup.7 HPβCD is hydroxypropylcyclodexdrin having 7.4 hydroxypropyl       groups per molecule of CD.                                               

                  TABLE 3                                                         ______________________________________                                        Effect of Concentration of Lipophilic Oligosaccharide Antibiotic              NMG Salt and Molar ratio of NMG to Antibiotic upon                            Adverse Reaction Syndrome.sup.1 in Mice                                       Drug of Formula III                                                                        % ARS.sup.1 at the following doses (MPK.sup.2):                  (Drug Concentration)                                                                       50     100     200    300   400                                  ______________________________________                                        III.sup.3 : 2NMG                                                                           --     --      50     --    --                                   (10 mg/ml)                                                                    III: 2NMG    --      20     100    --    --                                   (20 mg/ml)                                                                    III: 2NMG    70     100     --     --    --                                   (50 mg/ml)                                                                    III: 3NMG    --     --      0      0     20                                   (20 mg/ml)                                                                    III: 3NMG    --     --      0      15    40                                   (40 mg/ml)                                                                    III: 3NMG    --     --      100    --    --                                   (80 mg/ml)                                                                    III: 5NMG    --     --      0      0     --                                   (20 mg/ml)                                                                    III: 5NMG    --     --      0      --    40                                   (50 mg/ml)                                                                    III: 9NMG    --     --      0      0      0                                   (20 mg/ml)                                                                    III: 9NMG    --     --      0      0      0                                   (40 mg/ml)                                                                    III: 9NMG    --     --      0      --     0                                   (80 mg/ml)                                                                    ______________________________________                                         Footnotes to Table 3                                                          .sup.1 Adverse Reaction Syndrome Symptoms observed in mice (groups of 5 t     10, CF1, average weight 20 g, Harlan Sprague Dawley, fasted 18 hours)         within 2 minutes after IV injection. (single dose)                            .sup.2 MPK is mg of the drug of Formula III per kg of body weight.            .sup.3 III is everninomicintype antibiotic compound represented by Formul     III.                                                                     

Perhaps even more surprisingly, we observed that the Minimum InhibitoryConcentrations ("MIC") in the in vitro models, and the 50% protectivedose ("PD₅₀ ") values in an in vivo mouse protection model, of thecomplex of 3 to 6 moles of hydroxypropyl-β-cyclodextrin "HPβCD" incombination with one mole of the compound of Formula III and 2 or 3moles of NMG were essentially the same as the MICs and PD₅₀ values forthe compound of Formula III and for those of the NMG salt of thecompound of Formula III with HPβCD in said models. The protein bindingvalues for the complex of 2 or 3 moles of NMG per of the compound ofFormula III with 6 moles of HPβCD remained-at 96-98% of the bindingvalues that we observed for the salt of the compound of Formula III with2 moles of NMG.

Thus, we have surprisingly discovered pharmaceutically acceptablecompositions of matter containing a lipophilic oligosaccharideantibiotic which allows effective delivery of such antibiotic to theserum of an animal such as a mammal especially a man afflicted with abacterial infection susceptible to treatment by such lipophilicoligosaccharide antibiotic of Formulas I, II and III.

The 2-hydroxypropyl derivatives of α-, β- and γ-cyclodextrin useful inthe present invention have about 2 to 11 hydroxypropyl groups permolecule of cyclodextrin and are readily prepared by reacting one of α,β- or γ-cyclodextrin with 1,2-propylene oxide in the presence of base ina manner to yield multicomponent, amorphous mixtures such as describedby J. Pitha in U.S. Pat. No. 4,727,064, J. Pitha, et al., inInternational Journal of Pharmaceutics (1986), 29. 73-82 or by Muller inU.S. Pat. No. 4,870,060. The self condensation products of propyleneoxide are removed and the degree of substitution, i.e., number of thehydroxypropyl groups per molecule of cyclodextrin are convenientlydetermined by proton nuclear magnetic resonance and/or mass spectroscopyin accordance with the methods described by Pitha in U.S. Pat. No.4,727,064 and International Journal of Pharmaceutics (1986) 29. 73-82 orC. T. Ras, et al. in Pharmaceutical Research (1990) 7, (No. 6) 612-615.The 2-hydroxypropyl-α-, -β- and -γ-cyclodextrins having about 2 to 15,preferably about 3 to 9, more preferably about 5-7.5 hydroxypropylgroups per molecule of α-, -β- and -γ-cyclodextrin are readily preparedby conventional procedures and are also available commercially fromCyclolab, a wholly owned subsidary of CHINOIN, Pharmaceutical andChemical Works. Ltd., 1325 Budapest, Hungary; Walker-Chemie GmbH,Division L. Biotechnology Prinzregentenstraβe 22 D-8000, Munchen 22 WestGermany; American Maize, Hammond, Ind.; Pharmatec Inc, P.O. Box 730,Alachua Fla. 32615 and Janssen, Biotech N.V. Lammerdries 55, B-2430Olen, Belgium.

The amounts of such hydroxypropyl-α-, -β- and -γ-cyclodextrinssufficient to achieve efficacious delivery of the lipophilicoligosaccharide antibiotic to the serum of an animal, especially a humanbeing, without causing the adverse reaction syndrome upon injection of apharmaceutical composition containing a lipophilic oligosaccharideantibiotic of Formula I, II or III, at least a stoichiometric amount ofa selected base and an amount of a hydroxypropyl-α-, -β- or-γ-cyclodextrin in accordance with this invention are given in theparagraph hereinbelow.

Such an amount of hydroxypropyl-β-cyclodextrin ("HP-α-CD") is about 5 to15 moles of HP-α-CD per mole of a lipophilic oligosaccharide antibioticof Formula I. Such an amount-of hydroxypropyl-β-cyclodextrin (HPβ-CD) isabout 1-9, preferably about 2-6, moles of HP-β-CD per mole of alipophilic oligosacchide antibiotic of Formula II or III; such an amountof hydroxypropyl-γ-cyclodextrin ("HP-γ-CD") is about 2-8 moles,preferably about 3-5 moles of HP-γ-CD per mole of a lipophilicoligosaccharide antibiotic of Formula I. The salts of the lipophilicoligosaccharide antibiotic represented by Formula III are uniquelysuitable for forming complete solutions in water with a broad range ofhydroxypropyl-α-, -β- and -γ-cyclodextrins having from about 2 to 15hydroxypropyl groups per molecule of HP-α-CD or HP-β-CD or HP-γ-CD. Asshown in Table 4, a broad range of HP-β-CDs containing from 3.9 to 7.5hydroxypropyl groups per HP-β-CD molecule essentially prevented adversereaction syndrome upon parenteral injection into a mouse model of acomplex of one mole of the compound of Formula III and 2 moles of NMGand 6 moles of HP-β-CD.

                  TABLE 4                                                         ______________________________________                                        Adverse Reaction Syndrome.sup.1 upon Parenteral Injection (800                MPK, 80 mg of the compound of Formula III per ml) into Mice                   of an Aqueous Solution Complexes of One Mole of the Compound                  of Formula III and Two Moles of NMG and Six Moles of                          HP-β-CD having from 3.9 to 7.5 Hydroxypropyl Groups                      Number of Hydroxypropyl Groups                                                                   % Adverse Reaction                                         per molecule of HP-β-CD                                                                     Syndrome                                                   ______________________________________                                        No HP-β-CD    90                                                         7.4                0                                                          7.5                0                                                          7.4                0                                                          7.4                0                                                          7.2                0                                                          6.9                0                                                          6.7                10                                                         6.3                0                                                          6.2                0                                                          5.2                0                                                          4.5                0                                                          4.4                0                                                          4.1                10                                                         3.9                0                                                          ______________________________________                                         .sup.1 Adverse Reaction Syndrome observed in mice (groups of 10 CF1,          average weight 20 g, Harlan Sprague Dawley, fasted 18 hours) within 2         minutes after IV injection. (single dose)                                

In the course of development of the compositions of matter of thisinvention, we prepared such compositions by addition of a lipophilicoligosaccharide antibiotic, e.g., the compound represented by FormulaIII to at least a stoichiometric amount, e.g., about 2 to 3 moles of thepreferred base, N-methyl glucamine and a sufficient amount ofdimethylsulfoxide or glycerol or of sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivatives, e.g., polysorbate 80 or Tween 80, or adextran such as Dextrans 40 or 70 brands of polysaccharides [produced byaction of bacteria growing on sucrose substrate containing a backbone ofD-glucose units linked predominantly α-D (1→6). Dextran 40 and 70(average molecular weight of 40,000 and 70,000, respectively) arepolysaccharides produced by the action of L. mesenteroxides on sucrose;such sufficient amounts of dimethyl sulfoxide, glycerol, polysorbate 80or a dextran were found to achieve efficacious delivery of saidlipophilic oligosaccharide antibiotics to the serum of an animal whilesimultaneously reducing or avoiding occurrence of adverse reactionsyndrome. Reference is made to the results in Table 2. Increasing theamount of DMSO and glycerol [to 10 weight percent] reduced theoccurrence of adverse reaction syndrome; the sufficient amounts of DMSOand glycerol are about 5 to 10 weight percent and about 10 weightpercent, respectively. Increasing the amount Dextrans 40 and 70 alsoreduced the occurrence of adverse reaction syndrom; such sufficientamounts of Dextran 40 and Dextran 70 are about 2-10, and at least about6 weight percent respectively. Amounts of Tween 80 from about 0.1 weightpercent to about 1 weight percent increased occurrence of adversereaction syndrome but amounts of Tween 80 greater than 1 weight percentdecreased occurrence of adverse reaction syndrome while amounts of Tween80 greater than about 2 to about 3 weight percent resulted in almostcomplete avoidance of adverse reaction syndrome. Amounts of Tween 80greater than 2 to about 3 weight percent are Considered sufficient (allweight percents are based the total weight of the composition). However,the use of hydroxyl propyl-α-, -β- or γ-cyclodextrins is preferred forthe pharmaceutically acceptable compositions of this invention. SeeTable 2 hereinabove.

The bases found suitable for use in the present invention are thosewhich form pharmaceutically acceptable salts of the lipophilicoligosaccharide antibiotics of Formulas I, II or III and includesuitable organic and inorganic bases. Suitable organic bases includeprimary, secondary and tertiary alkyl amines, alkanolamines, aromaticamines, alkylaromatic amines and cyclic amines. Exemplary organic aminesinclude the pharmaceutically acceptable bases selected formchloroprocaine, procaine, piperazine, glucamine, N-methylglucamine,N,N-dimethyl glucamine ethylenediamine. diethanolamine,diisopropylamine, diethylamine, N-benzyl-2-phenylethylamine,N-N'-dibenzylethylenediamine, choline, clemizole,tris(hydroxymethyl)aminomethane, or D-glucosamine. The preferred organicbases include N-methyl glucamine ("NMG"), diethanolamine, andtris(hydroxymethyl) aminomethane ("TRIS"). Use of NMG in this inventionis more preferred. See Tables 2 and 3. The suitable inorganic basesinclude alkali metal hydroxides such as sodium hydroxide. The basesfound useful in the preparation of compositions of matter of the presentinvention produce aqueous solutions having a pH of at least about 9.3.Lysine forms aqueous solutions having a pH of less than 9.3 and thuslysine is not a suitable base for the present invention. Divalent metalhydroxides such as the alkaline earth hydroxides, calcium hydroxide andbarium hydroxide did not form aqueous solutions of the lipophilicoliogosaccharide antibiotics of Formulas I, II or III in the presence of6 moles of HP-β-CD having a pH of at least about 9.3 and wereunacceptable as bases for use in the present invention.

The term "at least about a stoichiometric amount" as used herein inreference to the bases Useful in this invention means the amount of baseneeded to substantially completely react with (i.e. result in more than99% complete reaction) the acidic phenolic hydrogens of the lipophilicoligosaccharides antibiotics of Formulas I, II, III having one or two orthree phenolic hydrogens. (Compound of Formula III has three phenolichydrogens of which only two are acidic). For the compounds of Formulasand II wherein R₅ =H, there is only one phenolic acidic hydrogen permolecule and the stoichiometric amount of the pharmaceuticallyacceptable bases of this invention is at least about one mole of suchbase up to 12 moles of such bases. For the compounds represented byFormula I and II wherein ##STR8## and for the compounds of Formula IIIthere are two acidic phenolic hydrogens per mole of such compounds, thestoichiometric amount of base required to completely react with the twoacidic phenolic hydrogens is at least 2 up to about 12 moles of thepharmaceutically acceptable bases useful in this invention. For thepreferred lipophilic oligosaccharide antibiotics of Formula I, and IIwherein ##STR9## and those of Formula III, it is preferred to use about2 to 6 moles, and it is more preferred to use about 2.0 to 3.5 moles andmost preferred to use about 2 to about 3 moles of a pharmaceuticallyacceptable base such as NMG to maintain the pH of an aqueous solutionthereof at a value of about 9.3 as opposed to solutions having a higherpH, and which solutions were highly buffered widen 6-12 moles of NMGwere used.

The term "lipophilic oligosaccharide antibiotic" as used herein meansselected lipophilic members of the orthosomycin family of antibiotics,more particularly fiambamycin, the everninomicins, everninomicin-typeantibiotics, curamycin and the avilamycin A-N antibiotics

Flambamycin, a lipophilic oligosaccharide antibiotic produced byStreptomyces hygroscoicus DS 23230, whose structural Formula is that ofFormula I wherein R₁ =R₅ =H, Y=OH, R₂ =COCH(CH₃)₂, R₃ =R₆ =R₇ =R₈ =R₉=CH₃, R₄ =COCH₃ and W=Z═Cl is disclosed by W. D. Ollis in Tetrahedron,(1979), 35, 105-127.

Curamycin A is a flambamycin antibiotic (having a structural Formularepresented by Formula I wherein R₁, R₃, R₄, R₅, R₆, R₇, R₈, R₉, W and Zare the same as for flambamycin except R₂ =COCH₃ and Y=H. See O. L.Galamarine et al. Tetrahedron (1961), 15, 76 and V,. Deulofer et al.,Anales de Quimica (1972), 68, 789.

Avilamycin A-N antibiotics are lipophilic oligosaccharide antibioticsisolated from an antibiotic complex produced by cultures of the organismStreptomyces viridochromogenes, NRRL 2860. See J. L. Mertz et al. TheJournal of Antibiotics (July 1986) Vol. 39 (No. 7) 877-887. Thestructural Formulas for the avilamycin A-N antibiotics are representedby Formula I wherein, R₁ =R₅ =H, Y=H, R₂ =COCH(CH₃)₂, COCH₃, CO(CH₂)₃CH₃, COCH₂ CH₃ or H, R₃ =CH₃, R₄ =COCH₃, CH(OH)CH₃ or CHO and R₆ =CH₃ orH; R₇ =CH₃ or H; R₈ =CH₃, CH₂ OH or H; R₉ =CH₃ or H and W=H or Cl andZ═Cl.

The everninomicin antibiotics useful in this invention include theeverninomicins B, C and D isolated from the antibiotic complex producedby the organism, Micromonospora carbonacea var. carbonacea NRRL 2972 anda variety thereof M. carbonacea var. aurantiaca NRRL 2997 as describedin U.S. Pat. No. 3,499,078. The everninomicin derivatives having anitroso, hydroxylamino or amino moiety in place of the nitro moiety ineverninomicins B, C and D may be obtained by reduction of the nitromoiety in everninomicins B, C and D in accordance with the procedures ofU.S. Pat. No. 4,006,225. A preferred everninomicin isN-acetylaminoeverninomicin-D and is represented by Formula II whereinX═NHCOCH₃, Y=H; R₄ =CH(OCH₃)(CH₃); R₃ =R₅ =H and R₂ =CH₃.N-acetylaminoeverninomicin-D and its di N-methylglucamine salt may beprepared by the procedures of U.S. Pat. No. 4,129,720 which disclosesreduction of the nitro moiety of everninomicins B, C and D to producethe amino derivatives which are subsequently convened into the N-acyle.g. N-acetyl, N-alkyl, e.g. NH(C₂ H₅), or N,N-dialkyl, e.g. N(C₂ H₅)₂,derivatives. The preparation of the N-acyl-N-hydroxylamino everninomicinB, C and D derivatives and pharmaceutically acceptable salts thereof arealso described. The preparation of everninomicin 7 represented byFormula II wherein X=OH, Y=H, R₄ =CH(OCH₃)(CH₃), R₅ =H and R₂ =CH₃ isdisclosed by A. K. Ganguly et al. in J. Chem. Soc., Chem. Corem. 1980,56-58.

The "everninomicin-type" antibiotics are those lipophilicoligosaccharide antibiotics represented by Formula II wherein X=NO₂, NONH₂, OH, NHCOCH₃, NHC₂ H₅, N(C₂ H₅)₂, NHOH or H, Y=OH, R₂ =CH₃ or H; R₃=H, R₄ =CH(OCH₃)(CH₃) or H and ##STR10##

The compounds of Formula II wherein X=NO₂ or NH₂, Y=OH R₂ =R₃ =R₄ =H,and ##STR11## are isolated from an antibiotic 13-384 complex produced byfermentation of the organism Micromonospora carbonacea var. africana,NRRL 15099, ATCC 39149. Antibiotic components 1 (Formula II, X=NO₂ andY, R₂, R₃, R₄ and R₅ are each defined as hereinabove in reference toantibiotic 13-384) and 5 (Formula II, X=NH₂ and Y, R₂, R₃, R₄, and R₅are as defined as hereinabove in reference to antibiotic 13-384)disclosed n U.S. Pat. No. 4,597,968 and 4,735,903 have the structuralFormulas disclosed by AK Ganguly et al. in Heterocycles (1984) Vol. 28(No. 1) p 83-88. The everninomicin-type antibiotics of Formula IIwherein X=H, NHOH, NHCOCH₃ and acyl and alkyl derivatives thereof aredescribed in U.S. Pat. No. 4,622,314 and 4,767,748.

The preferred compositions of matter of this invention include compoundsof Formula II wherein R₃ =H,

    __________________________________________________________________________    and  X   Y  R.sub.4    R.sub.5    R.sub.2                                     __________________________________________________________________________    NO.sub.2 OH CH(OCH.sub.3)(CH.sub.3)                                                                  H          CH.sub.3                                    OH       H  "          "          "                                           NO.sub.2 H  H          "          "                                           NO.sub.2 H  CH(OCH.sub.3)(CH.sub.3)                                                                  "          "                                           NHCOCH.sub.3                                                                           H  "          "          "                                           NO.sub.2 OH H                                                                                         ##STR12## H                                           H        "  "          "          "                                           NHOH     "  "          "          "                                           NHCOCH.sub.3                                                                           "  "          "          "                                           NH.sub.2 "  "          "          "                                           NHC.sub.2 H.sub.5                                                                      "  "          "          "                                           N(C.sub.2 H.sub.5).sub.2                                                               "  "          "          "                                           __________________________________________________________________________

The most preferred everninomicin-type antibiotic is named56-deacetyl-57-demethyl-45-O-de(2-methyl-1-oxopropyl)-12-O-(2,3,6-trideoxy-3-C-methyl-4-O-methyl-3-nitro-α-L-arabino-hexopyransoyl)-flambamycin56-(2,4-dihydroxy-6-methylbenzoate) having the molecular Formula of: C₇₀H₉₇ NO₃₈ Cl₂ and the molecular weight of 1629 and is represented byFormula III.

The preferred compound of the Formula III may be obtained byfermentation of Micromonospora carbonacea var. africana NRRL 15099, ATCC39149 or, more preferably, by an improved strain thereof, obtained ashereinafter described.

Utilizing the strain SCC 1413 of the culture NRRL 15099, ATCC 39149, thepreferred compound of the Formula III may suitably be obtained by theprocedures outlined in Example 1 of U.S. Pat. No. 4,597,968. In aspecific example, in accordance with this procedure, the initial stageinoculum for the fermentation of strain SCC 1413 was prepared bytransferring 2.5 ml of a frozen whole broth of 50 ml of the germinationmedium in 250 ml Erlenmeyer flasks. The germination medium consisted ofbeef extract, 0.3%; tryptone, 0.5%; dextrose, 0.1%; potato starch,2.43%; yeast extract, 0.5%; and calcium carbonate, 0.2%. The pH of themedium was adjusted to 7.5 prior to sterilization. The flasks wereincubated at 30° C. on a gyratory shaker at 250 r.p.m. for 48 hours. Forthe second stage germination, 2 liter Erlenmeyer flasks containing 500ml of the same medium were inoculated with a 5% volume of the firststage germination. The conditions for incubation were the same asbefore. A third inoculum stage was employed for all stirred tankfermentations and was prepared by a 24 hour incubation of the cultureunder the same conditions as employed for the second stage.

Ten liter fermentations were initially carried out in 14 liter NBSLaboratory Fermentors in a fermentation medium containing yeast extract,0.5%; casein hydrolysate, 0.5%; cerelose, 1%; soluble starch, 2.0%;calcium carbonate, 0.4%; and cobalt chloride, 0.24 mg %. The pH of themedium was adjusted to 6.7 before sterilization and to 7.0 beforeinoculation. The third stage inoculum (22.5%) was used to initiate thefermentation which was conducted at 30° C. with 0.35 vvm of air and 350rpm agitation.

During the course of the fermentation, antibiotic production wasmonitored every 24 hours by bioassay of the whole broth againstStaphylococus aureus 209 P (pH of the agar, 7.0) and Escherichia coliATCC 10536 (pH of the agar, 8.0). The growth of the producing organism(packed cell volume ), pH and dissolved oxygen levels were alsodetermined either intermittently or continuously. The course of atypical 10 liter tank fermentation is illustrated in FIG. 1.

We have developed an improved strain from SCC1413, NRRL 15099, ATCC39149 using standard mutagenesis agents and obtained strains producingimproved yields of the preferred everninomicin-type antibiotic compoundof the Formula III. In a specific example, the parent strain SCC 1413,NRRL 15099, ATCC 39149 was exposed to an amount of the mutagenesisagent, N-nitrosoguanidine (NTG) sufficient to kill 90% of a culture ofSCC 1413, ATCC 39149, NRRL 15099. Fifteen hundred surviving isolateswere examined for-enhanced biological activity against S. aureus and E.coli to determine which isolates exhibited improved production of thedesired antibiotic of Formula III. The test procedure employed todetermine enhanced activity was as follows: Single colony isolates weregerminated in test tubes containing 10 ml of germination media ofExample 1 of U.S. Pat. No. 4,597,968 and shaken at 250 r.p.m. on agyratory shaker at 30° C. for 48 hours. Fermentation studies wereinitiated by transferring 2.5 ml of the seed to 250 ml Erlenmeyer flaskscontaining 50 ml of fermentation media and incubating at 30° C. for 96hours at 250 r.p.m. on a gyratory shaker. The antibiotic obtainedfollowing fermentation was then assayed for improved antibioticproduction by assessing the activity against S. aureus and E. coli andisolates giving improved yields of the desired antibiotic wereidentified. The results for a representative improved isolate,designated herein as strain SCC 1631, are given in Table 5.

The foregoing strain-development procedure was repeated by subjectingthe representative improved isolate, SCC 1631, to a further exposure toNTG, again in an amount sufficient to kill 90% of the cultures, followedby selection of the isolates on agar plates containing 150 μg/mL ofeverninomicin D. Isolates giving enhanced production of the desiredantibiotic were again selected by assessing biological activity thereofagainst S. aureu and E. coli. One such isolate, herein designated strainSCC 1756, was then utilized to produce the preferred antibiotic ofFormula III.

Further, NTG mutagenesis of SCC 1756 yielded our current productionstrain, SCC 2146.

In the foregoing mutation procedures, the protocols for both studieswere as previously described hereinabove. For the latter two mutationstudies, fermentation broths were extracted with ethyl acetate and theconcentrates were chromatographer on Whatman LKGDF thin layer plates ina solvent system consisting of chloroform:methanol (9:1 v/v) followed bybioautography against S. aureus and E. coli to confirm the production ofall components of the antibiotic complex. To follow increased titers ofthe compound of Formula III, thin layer plates were examined by usingthe Shimadzu CS-930 TLC plate scanner and quantitating the higherproducing extracts by using HPLC. Combined titers are defined as the sumof the compound of Formula III (antibiotic 13-384, component 1 of U.S.Pat. No. 4,597,968) and the nitroso analog of said component 1, i.e.,antibiotic 13-384, component 1a.

Early observations indicated that although the parent strain SCC 1413grew rapidly at 34° C., antibiotic production was optimal if thetemperature was lower. This phenomenon was investigated as a means offermentation optimization. Results of the temperature study indicatedthat optimal production was obtained when the temperature was loweredfrom 34° C. to 30° C. after 24 hours of incubation. All subsequent workin stirred tanks followed the protocol of incubating the fermentation at34° C. for 24 hours followed by lowering the temperature to 30° C. forthe duration of the fermentation run.

Media studies were conducted in conjunction with the isolation of theimproved production strains. Carbon and nitrogen source substitutionswere investigated as well as the addition of minerals and other complexnutrients. Replacement of casein hydrolysate by either meat or fishpeptone and substituting potato dextrin (PDP 650) for soluble starchenhanced antibiotic production using strains SCC 1413 and SCC 1631.Subsequent enhancements in the production of the compound of Formula IIIwere observed with the addition of corn steep liquor and nickel (II)chloride in studies with strain SCC 1756. The current productionfermentation media (4I+ 1/2 Ni) optimized for the compound of FormulaIII contains glucose, 2.2 weight %; PDP 650 dextrin, 4.0 weight %; yeastextract, 0.5 weight %; meat peptone, 0.6 weight %; corn steep liquor,0.5% vol., nickel chloride, 2.5×10⁻⁶ M; and calcium carbonate, 0.4weight %. The pH of the medium was adjusted to 6.7 before the additionof calcium carbonate. Table 6 shows a comparison of the titers forstrains SCC 1413, SCC 1631, SCC 1756 and SCC 2146 obtained in shakeflask studies (50 ml of the current production medium in 250 mlerlenmeyer flasks, at 30° C., for 96 hours, at 300 r.p.m.). The markedtiter improvement (15 fold over the original parent, SCC 1413)is clearlydemonstrated. Titers of 555-750 ug/ml (sum of the compound of FormulaIII and the nitroso derivative thereof) have been achieved in 100 literfermentations using the current production medium with our bestproduction strain. SCC 2146 (Table 7).

                                      TABLE 5                                     __________________________________________________________________________    Comparison of Strains SCC 1413 and SCC 1631 in Fermentations                  Showing, Zones of Inhibition (mm) on Agar Plates.sup.1                        TEST 1               TEST 2                                                   Strain                                                                              S. aureus pH 7                                                                       E. coli pH 8                                                                          S. aureus pH 7                                                                          E. coli pH 8                                   SCC  Undil.                                                                             1:20                                                                             Undil.  Undil.                                                                             1:20 Undil.                                         __________________________________________________________________________    1631 28.7, 28.7                                                                         22.0                                                                             20, 17.5                                                                              28.1, 28.8                                                                         23.3, 23.1                                                                         14.8 C.sup.2, 15.0 C.sup.2                     1413 28.7, 28.7                                                                         19.0                                                                             12 H.sup.3, 12H.sup.3                                                                 23.8, 23.1                                                                         20.5, 19.8                                                                         12 H.sup.3, 12 H.sup.3                         __________________________________________________________________________     .sup.1 Duplicate Determinations Where Appropriate                             .sup.2 Clear Zone                                                             .sup.3 Hazy Zone                                                         

                  TABLE 6                                                         ______________________________________                                        Flask Comparison of SCC's 1413, 1631, 1756 and 2146                           Strains of Micromonospore Carbonacea var africana NRRL                        15099, ATCC 39149                                                             Titer of the compound of Formula III and Nitroso Analog (1A)                  Thereof (μg/ml)                                                            Culture 1 (NO.sub.2)                                                                              1a (NO)  combined (1 + 1a)                                ______________________________________                                        SCC 1413                                                                               5           3        8                                               SCC 1631                                                                              14           4       18                                               SCC 1756                                                                              17          16       33                                               SCC 2146                                                                              39          85       124                                              ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        100 Liter Fermentations of SCC 2146                                           Titer of Formula III and the NO Analog (1A) Thereof (μg/ml)                Media    1 (NO.sub.2).sup.1                                                                       1a (NO.sup.2)                                                                          combined (1 + 1a)                                ______________________________________                                        4I       105        315      420                                              4I       135        170      305                                              4I +  1/2Ni.sup.3                                                                       55        500      555                                              4I + Ni.sup.4                                                                          150        575      725                                              4I +  1/2Ni.sup.3                                                                      100        650      750                                              4I +  1/2Ni.sup.3                                                                      130        470      600                                              ______________________________________                                         Footnotes to Table 7                                                          .sup.1 The everninomicintype antibiotic of Formula III.                       .sup.2 The Nitroso analog of the antibiotic of Formula III.                   .sup.3 Nickel concentration (1/2 Ni) = 2.5 × 10.sup.-6 M.               4Nickel concentration Ni = 5 × 10.sup.-6 M                         

The isolation of the lipophilic oligosaccharide antibiotic complexcontaining the compound of Formula III and the nitroso analog thereofwas accomplished by use of the procedures of Example 1C of U.S. Pat. No.4,597,968. The fermentation broth was adjusted to pH 7 and extractedtwice with a volume of ethyl acetate two times the volume of thefermentation broth. The combined ethyl acetate extracts wereconcentrated and the amounts of the compound of Formula III and thenitroso analog thereof were determined by HPLC, The nitroso analog wasconvened into the nitro compound of Formula III by use of an oxidizingagent such as tertiary butyl hydroperoxide (t-BuO₂ H) with vanadylacetylacetonate dissolved in an aprotic organic solvent at roomtemperature. The course of the reaction was monitored by, for example,HPLC. The reaction mixture was quenched with trialkylphosphite and thecrude product was purified by standard chromatographic techniques. e.g.silica gel column chromatography (acetone/CH₂ Cl₂) or a columncontaining a polyhydroxyvinyl polymer such as Fractogel (Toyo Pearl)available from Toyo Haas, Philadelphia, Pa.

The pharmaceutically acceptable conposition of matter of this inventionmay contain, in addition to (a) an antibiotic of Formula I, Il, III ,(b) a base capable of forming a pharmaceutically acceptable salt of suchantibiotics and (c) an a specified amount of, for example,hydroxypropyl-α-, -β-, or-γ-cyclodextrin having 2 to 15 hydroxypropylgroups per molecules of cyciodextrin, about 0 to about 6.0 weightpercent (basis on antibiotic of Formula I, II, III) of apharmaceutically acceptable non-ionic surfactant. The preferredpharmaceutically acceptable non-ionic surfactant, when used, is thesorbitan mono-9-octadecenoate poly (oxy-1,2-ethanediyl) derivative suchas Tween-80, but any other non -ionic surfactant which producespharmaceutically acceptable compositions, i.e., compositions which whendissolved in a pharmaceutically acceptable carrier are substantiallyfree of haze, cloudiness and particulate matter as measured by standardanalytical techniques, e.g., nephelometry. Particularly preferredcompositions of matter of this invention contain 2.85 to 5.70% by weightof Tween-80 and a antibiotic compound of Formula III. "The preferredpharmaceutical composition of this invention contains the following: (a)the lipophilic oligosaccharide antibiotic represented by Formula III,(b) a base capable of forming a pharmaceutically acceptable salt withlypophilic oligosacharide antibiotic of Formula III and (c)hydoxypropyl-α-, β- or-γcyclodextrin having about 2 to about 15hydroxypropyl groups per molecule of said α-β and γ-cyclodextrin andwherein the molar ratio of (a):(b):(c) is 1:2-3:1-6."

BIOLOGICAL ACTIVITIES

We have surprisingly found that the preferred composition of matter ofthis invention, comprising one mole of the compound represented byFormula III 2 moles of NMG and 6 moles of 2-hydroxypropyl-βcyclodextrinhaving 7.4 hydroxypropyl groups per molecule of β-cyclodextrin, hassubstantially the same geometric mean MICs (GMM) against variousbacteria, and substantially the same serum protein binding values as thecompound of Formula III per se. It is expected that all the compositionsof matter of this invention will behave similarly.

The in vitro antibacterial activity tests were performed viaconventional agar dilution methods in Mueller--Hinton agar. The GMMs forthe above-listed preferred composition of matter of this invention andfor the compound of Formula III were determined against vadous bacteria,e.g., gram positive and gram negative bacteria. The term "susceptiblegram positive and gram negative bacterial infections " means a broadrange of gram positive bacterial infections. e.g., methicillin-resistantand methicillinsus-ceptible staphylococci, various strains ofstreptococci and enterococci and some gram negative bacterialinfections, e.g., E. coli Klebsiella, Salmonella and Pseudomonas. Thecompound of Formula III had excellent activity (10-fold more potent thanvancomycin) against both methicillin-resistant staphylococci (GMM, 0.1μg/ml) and methicillin-susceptible staphylococci (GMM, 0.5 μg/ml). Thecompound of Formula III also had good activity (2-fold more potent thanvancomycin) against Enterococcus faecalis (GMM, 0.49 μg/ml) and goodactivity (MICs, ≦0.5 μg/ml) against various strains of streptococci andenterococci resistant to vancomycin (MICs, ≧128 μg/ml). The compound ofFormula III was very active against Borrelia burgdorferi (MICs, ≦0.49μg/ml) and Legionella pneumophila and L. longbeacheae (MICs 2.5 μg/ml)but was only slightly active against gram-negative bacteria (GMM, ≧760μg/ml), Trichomonas vaginalis (MICs, ≧192 μg/ml) and Mycoplasma sp.(MICs 200 μg/ml). No cross resistance with other antibiotics wasobserved.

The compound of Formula III had moderate bactericidal activity againstvarious clinical and laboratory strains of staphylococci. Thebactericidal activity of the compound of Formula III againststaphylococci and enterococci was similar to that of vancomycin. Thecompound of Formula III had good activity against staphylococci in mice(PD₅₀ range 0.5 to 25.0 mg/kg), similar to that of vancomycin (0.7 to28.5 mg/kg).

Following IV administration (30 mg/kg) of the compound of Formula IIIand 2 molecules of NMG, high serum levels were seen in rats (peak about90 ug/ml) with a long serum beta half life.

The pharmaceutically acceptable compositions of matter of this inventionare expected to be active against the above-listed susceptible bacteriaas well as against. spirochetes including Treponema pallidum, anaerobesincluding Clostridium difficile as well as against Pneumocystis.Toxoplasma. protozoa and helminths.

Based on the activity of the compound of Formula III against borreliaburgdorferi and Legionella pneumophila and L, longbeacheae, expect thatthe compositions of matter containing the compound of Formula-III willexhibit activity in a human model against Lyme disease and iegionaire'sdisease.

The present invention provides a method of treating or preventingsusceptible gram-posititive and gram-negative bacterial infections inanimals by administering to such animals especially man afflicted withsuch infections an amount of a pharmaceutical composition of thecompositions of matter of this invention and a pharmaceuticallyacceptable carrier therefor.

The compositions of matter of this invention may be combined with anypharmaceutically acceptable carrier, e.g., sterilized water, aqueousethanol, vegetable oils, or polyols, e.g., polyethylene glycols andpropylene glycol and administered orally, parenterally or topically in avalet of formulations. The use of sterilized water as a carrier ispreferred. The sterilized water may optionally contain pharmaceuticallyacceptable substances, e.g. sodium chloride, potassium nitrate, glucose,mannitol, dextrose, sorbitol, xylitol or buffers such as phosphate, aCetate or citrate as well as preservatives.

The compositions of matter of this invention are prepared by admixing alipophilic oligosaccharide antibiotic of Formula I, II or III with atleast about a stoichiometric amount of a base capable of forming apharmaceutically acceptable salt thereof in a suitable solvent such aswater. and with a specified amount of, for example hydroxypropyl-α-, -β-or -γ-cyclodextrin having about 2 to 15 hydroxypropyl groups permolecule of cyclodextrin. The order of admixing is not critical, butpreferably an aqueous solution of the specific cyclodextrin is admixedwith the base or alternatively it may be added after the base is admixedwith the lipophilic oligosaccharide antibiotic. The formation of theaqueous solutions may take place at a temperature between 15° and 35° C.The aqueous solution so formed is filtered to produce a clear aqueoussolution of the complex which may be evaporated or preferablyfreeze-dried to form the compositions of matter of this invention in theform of a lyophilized powder which is readily re-constituted by additionan amount of a pharmaceutically acceptable carrier such as water. Thepharmaceutically acceptable non-ionic surfactant e.g. Tween -80whenused, would be added to the aqueous solution before filtration andlyophilization. Alternatively, the aqueous solution may be frozen,thawed and thereafter filtered before use, e.g., as an IV formulation.It is a special feature of the present invention that the pharmaceuticalcompositions of the present invention form aqueous solutions and yetcontain less than about 20 weight percent, preferably less than 10 andmore preferably about 1.0 to 5.0 weight percent of an amount ofhydroxypropyl-α-, -β- or -γ-cyclodextrin. The discovery thatpharmaceutical compositions useful for safely and effectively deliveringlipophilic oligosaccharide antibiotics to the serum of animals afflictedwith susceptible bacterial infections, especially susceptible grampositive and gram negative bacterial infections, could be prepared byuse of less than 20 weight percent. of for examplehydroxypropyl-β-cyclodextrin is particularly surprising in view of theteachings of the Bodor U.S. Pat. No. 4,983,586 to use 20 to 50 weightpercent of hydroxypropyl-β-cyclodextrin to avoid gelling orprecipitation of drug and of the teaching of Pitha U.S. Pat. No.4,727,064 to use 40 to 60 weight percent of hydroxypropyl-β-cyclodextrinto solubilize various drugs including salts of retinoic acid.

For oral administration, the compositions of this invention may becompounded in the form of tablets, capsules, elixers or the like.Tablets and capsules may contain such excipients as starch or lactose;liquid forms may contain coloring or flavoring agents. Topicalspreparations may be in the form of creams, hydrophobic and hydrophylicointments, or aqueous, non-aqueous or emulsion-type lotions as well aspessaries or powders. Typical carriers for such formulations are water,oils, greases, polyesters and polyols. Parenteral formulations, e.g.,injectable dosage forms, are usually liquids such as solutions orsuspensions, with typical carriers being distilled water and salinesolution. Parenteral formulations are preferred. Intravenous (IV)formulations are more preferred.

The dose to be administered in any particular dosage form will dependupon various factors, such as the weight, age and sex of the animalespecially a mammal such as a human being being treated, thesusceptibility of the infecting organism to the lipophilicoligosaccharide antibiotic, the stage and severity of the infection.Generally, the dosage of the lipophilic oligosaccharide antibiotics ofFormula I, II or III administered is from about 1.0 mg to about 15 mgper kilogram of body weight, preferably about 5 mg per kilogram of bodyweight per day in divided dosages, the specified dosage being left tothe discretion of the practitioner; IV administration is preferred.

In treating certain patients with the compositions of this invention, itis possible to include other pharmaceutically active ingredients in thesame dosage unit.

EXAMPLES Example 1

A 100 liter fermentation of strain SCC 2146 of Micromonospora carbonaceavar. africana NRRL 15099, ATCC 39149 improved as described hereinabove,was conducted in accordance with the procedures of Example 1B of U.S.Pat. No. 4,597,968 except that the following production medium (4l+1/2Ni) was used and that the fermentation was conducted at 34° C. for 24hr followed by lowering the temperature to 30° C. for the duration ofthe fermentation run, i.e., for another 72 hr (total fermentation timeof 96 hr). Aeration and agitation rates were, 0.35 vvm and 350 rpm,respectively

    ______________________________________                                        Glucose               2.2% (weight)                                           PDP 650 dextrin       4.0% (weight)                                           Yeast Extract         0.5% (weight)                                           Meat Peptone          0.6% (weight)                                           Corn Steep Liquor     0.5% (volume)                                           Nickel Chloride       2.5 × 10.sup.-6 M                                 Calcium Carbonate     0.4% (by weight)                                        Tap Water q.s. to give                                                                              1000 ml                                                 ______________________________________                                    

B. Isolation

Extract the fermentation broth of Example 1A twice with 200 L of ethylacetate. Combine the ethyl acetate extracts and concentrate to provide aconcentrated antibiotic complex containing a mixture of the compound ofFormula III and the nitroso analog thereof (as determined by HPLC).

Example 2

A) To 919 g of antibiotic complex produced as described in Example I andcontaining 294 g (32%) of a mixture of 3.4 moles of the nitroso analogto one mole of the compound of Formula III dissolved in 4.6 L of ethylacetate, 68.8 g of NaHCO₃ and 2.98 g of vanadyl acetylacetonate 3M in2,2,4-trimethylpentane available-from Aldrich (0.06 eq); 394 mL of 3Mtbutylhydroperoxide was added to the so-formed mixture after a 1/2 hourperiod. Portions of 1.45 g (0.03 eq) of vanadyl acetylacetonate wereadded thereto at 0 and after 1 1/2, 2 1/2, 3 1/2 and 4 hours so that0.15 eq of vanadyl acetylacetonate was added over 4 hours. The reactionmixture was immersed in an ice bath, and 203 mL (0.5 eg) oftriethylphosphite (C₂ H₅ O)₃ P was added thereto. The so-formed reactionmixture was diluted with an equal amount of ethyl acetate while keepingthe temperature of the reaction mixture at ≦30° C. The diluted ethylacetate reaction mixture was washed twice with water. The aqueous layerswere salted and extracted with ethyl acetate, The combined organicextracts were dried over MgSO₄, filtered and concentrated. The so-formedresidue was dissolved in a minimum amount of acetone and precipitatedinto 7 L of 1:9 (v/v) ethyl ether/hexane. The residue was filtered andwashed with hexane dried under vacuum and heat to give 928 g containing30% (278 g) of the nitro compound of Formula III.

B) The residue of Example 2A was purified on 5 kg of silica gel in acolumn. The column was eluted with 12 liters of CH₂ Cl₂ containingsuccessively 10%, 20%, 25%, 30%, 35% (v/v) of acetone. The appropriatefractions were combined and concentrated at ≦35° C. The so-formedresidue was dissolved in acetone and precipitated into 10 parts of 10%ethyl ether/hexane. The product was filtered and dried under vacuumwithout heat. The main fraction contained 147.5 g of the compound ofFormula (98.7% pure). The other fractions contained crude product andwere subjected to repeated silica gel chromatography until at least a96-98% pure product was obtained. The structure was determined by NMRand MS and found to be consistent with that of Formula III.

Example 3A

An aqueous solution containing 23.97 mg of N-methyl glucamine ("NMG")and 570.90 mg of 2-hydroxypropyl -β-cyclodextrin ("HPβCD") having 7.4hydroxypropyl groups per molecule of HPβCD was prepared in 5 mL ofwater.

To this solution was added 100 mg of the compound of Formula III. Aftermild agitation, a homogeneous complex containing 20 mg per mL of thecompound of Formula III was formed. The molar ratios of the threecomponents were 1 mole of the compound of Formula III to 2 moles of"NMG" to 6 moles of HPβCD. The so-formed solution Was filtered through a0.45 μm membrane and freeze-dried and stored in a moisture-freeenvironment. For preparation of a pharmaceutical composition, apharmaceutically acceptable carrier such as water was added. Similarresults were obtained using HPβCD having 3.9 to 7.5 hydroxypropyl groupsper mole of β-cyclodextrin.

The formation of a clear aqueous solution of a homogeneous complexprepared in accordance with these procedure of this Example is verifiedby use of standard techniques, i.e., light scattering nephelometry andline width measurements in the proton NMR. The safe and effectiveadministration of the pharmaceutical composition of clear aqueoussolutions of the homogeneous complex prepared in accordance with theprocedures of this invention was tested in various in-vivo animal modelssuch are reported in Table 1-4.

Example 3B

The procedure of Example 3A is followed except that 1750 mg of HPβCDcontaining 7.4 hydroxpropyl groups per molecule was added to an aqueoussolution of 126 mg of NMG and 350 mg of the compound of Formula III. Themolar ratios of the three components in the homogeneous solutionso-formed were 1 mole of the compound of Formula III to 3 moles of NMGto 5 moles of HPβCD. To this solution were added 500 mg of granularmannitol, USP grade and 10 mg of Polysorbate-80 (Tween-80) NF. Theweight percent of Tween-80 is 2.85% basis the compound of Formula Theso-formed solution was filtered and freeze-dried as described in Example3A.

The freeze-dried composition is stored in vials in a moisturefreeenvironment. For preparation of a pharmaceutical composition suitablefor i.v. administration, 20 ml of sterile water is added.

Example 4

The following examples present the use of the NMR chemicalmicrotitration technique for the determination of the equilibriumconstants for complexing of the 2,4 dihydroxy 6-methyl phenyl ring(non-chloroaromatic) and for the 3,5-dichloro-2-methoxy-4-hydroxy6-methyl phenyl ring (dichloroaromatic ring) of NMG salt of the compoundof Formula III with 2-hydroxypropyl-β-cyclodextrin (HPCγCD) having 7.4and 3.4 hydroxypropyl groups per mole of HPβCD and with2-hydroxypropyl-γ-cyclodextrin ("HPγCD") having 4.4 hydroxypropyl groupsper mole of HPγCD. The measurements were made using a Varian XL 400 at400 mHz at 20° C.

A. A solution containing 10 mg of the compound of Formula III per ml ofD₂ O and 3 equivalents of N-methyl glucamine ("NMG") was prepared. Tothis solution was added mg portions of HPβCD having 7.4 hydroxypropylgroups per molecule of HPβCD. The change in chemical shifts of the 68methyl group in the Formula III and 54 methylene protons in Formula IIIwere measured as increasing amounts of HPβCD were added. The equilibriumconstants for 50% complexation were calculated using standardtechniques. The moles of HPβCD required to achieve 90% complexation orbinding at both aromatic rings were also determined. The moles of HPβCDrequired to achieve 90% complexation at both aromatic sites was 6.6; 2moles of HPβCD were required to achieve 50% complexation. Theequilibrium constants are reported in the following Table 8.

B. The procedures of Example 4A were followed except that HPβCD having3.4 hydroxypropyl groups per mole HPβCD was used. The results arereported in the following Table 8. The moles of HPβCD required toachieve 90% complexation at both aromatic sites was 6.6 mole.

C. The procedures of Example 4C were followed except that HPγCD having4.4 hydroxypropyl groups per mole of HPγCD was used. The moles of HPγCDrequired to achieve 90% complexation at both site was 4.0; 1.4 moleswere required to achieve 50% complexation. The results are reported inTable 8 below:

                                      TABLE 8                                     __________________________________________________________________________    Equilibrium Constants (Liters/Mole) for Complexes of 1 mole                   of Compound III: 3 moles of NMG with HPβCD and HPγC D              Complexation Site                                                             in compound III                                                                           HPβCD n.sup.1 = 7.4                                                               HPβCD n.sup.1 = 3.4                                                               HPγCD n.sup.2 = 4.4                       __________________________________________________________________________    Non-Chloro  183 ± 5                                                                             188 ± 5                                                                             355 ± 9                                      Aromatic Ring.sup.3                                                           .sup.3 DiChloro Aromatic.sup.4                                                            953 ± 18                                                                            983 ± 34                                                                            315 ± 28                                     Ring                                                                          __________________________________________________________________________     .sup.1 n = mean number of hydroxypropyl groups per molecule of HPβCD     .sup.2 n = mean number of hydropropyl groups per molecule of HPγCD.     .sup.3 NonChloro Aromatic Ring in III =                                       ##STR13##                                                                     .sup.4 The Dichloro Aromatic Ring in III =                                    ##STR14##                                                                

By use of the NMR chemical microtitration technique, the complexingamount of HPβCD having 7.4 hydroxypropyl groups per molecules of HPβCDrequired for one mole of the 3:1 NMG salt of the antibiotic of FormulaIII was determined to be 6.6 mole to achieve 90% complexation (at botharomatic groups in Formula III ) and to be 2 moles to achieve 50%complexation at both aromatic groups in Formula III.

What is claimed is:
 1. A composition comprising:(a) a lipophilicoligosaccharide antibiotic represented by Formula I; ##STR15## wherein##STR16## X is one of NO₂, NO, NH₂, NHCOCH₃, NHOH, NH(C₂ H₅), N(C₂ H₅)₂,OH or H;R₂ is one of CH₃, COCH(CH₃)₂, COCH₃, CO(CH₂)₃ CH₃, COCH₂ CH₃ orH; R₃ is CH₃ or H; R₄ is one of COCH₃, CH(OCH₃)(CH₃); CH(OH)CH₃, CHO, orH; ##STR17## R₆ is CH₃ or H; R₇ is CH₃ or H; R₈ is CH₃, CH₂ OH or H; R₉is CH₃ or H; Y is OH, H or CH₃ W is Cl or H; and Z is Cl or H; (b) atleast about a stoichiometric amount of a base capable of forming apharmaceutically acceptable salt with a lipophilic oligosaccharideantibiotic of Formula I; (c) a hydroxypropyl-α-β- or -γ-cyclodextrinwherein the average number of hydroxypropyl substituents on said -α, β-and γ-cyclodextrin is in the range of about 2 to about 15, and saidamount is sufficient to achieve efficacious delivery of said lipophilicoligosaccharide antibiotic to the serum of an animal whilesimultaneously avoiding occurrence of adverse reaction syndrome; and (d)0 to 6.0 by weight (basis an antibiotic of Formula I) of apharmaceutically acceptable non-ionic surfactant.
 2. The composition ofclaim 1 wherein the lipophilic oligosaccharide antibiotics representedby Formula I are selected from the group consisting of flambamycin, theeverninomicins, the everninomicin-type antibiotics, curamycin, and theavilamycin A-N antibiotics.
 3. A pharmaceutical composition for treatingsusceptible gram-positive and/or gram-negative bacterial infectionscomprising an antiinfective amount of a composition of claim 1 and apharmaceutically acceptable carrier therefor.
 4. The pharmaceuticalcomposition of claim 3 wherein the base is N-methylglucamine.
 5. Amethod of preventing adverse reaction syndrome in animals followingparenteral administration of a lipophilic oligosaccharide antibioticrepresented by Formula I of claim 1 while simultaneously delivering anantiinfective amount of a said antibiotic to said animal, said methodwhich comprises parenterally administering to said animal anantiinfective amount of a composition of claim 1 together with apharmaceutically acceptable carrier therefor.
 6. A compositioncomprising(a) a compound represented by the Formula II ##STR18## whereinX is one of NO₂, NO, NHOH, NH₂, NHCOCH₃, NH(C₂ H₅), N(C₂ H₅)₂,OH or H;Yis OH, H or CH₃ ; R₂ is H or CH₃ ; R₃ is H; R₄ is H or CH(OCH₃)(CH₃);and R₅ is H or ##STR19## (b) at least about a stoichiometric amount of abase capable of forming a pharmaceutically acceptable salt with acompound of Formula II; (c) an amount of hydroxypropyl-α-, -β- or-γ-cyclodextrin wherein the average number of hydroxypropyl substituentson said α, -β-and γ-cyclodextrin is in the range of about 2 to about 15,and said amount is sufficient to achieve efficacious delivery of saidlipophilic oligosaccharide antibiotic to the serum of an animal whilesimultaneously avoiding occurrence of adverse reaction syndrome; and (d)0 to 6.0% by weight (basis a compound of Formula II) of apharmaceutically acceptable non-ionic surfactant.
 7. A pharmaceuticalcomposition for treating susceptible gram-positive and/or gram-negativebacterial infections comprising an antiinfective amount of a compositionof matter of claim 6 and a pharmaceutically acceptable carrier therefor.8. The pharmaceutical composition of claim 7 wherein the base isN-methylglucamine.
 9. A method of preventing adverse reaction syndromein animals following parenteral administration of a lipophilicoligosaccharide antibiotic represented by Formula II of claim 6 whilesimultaneously delivering an antiinfective amount of a said antibioticto said animal, said method which comprises parenterally administeringto said animal an antiinfective amount of a composition of claim 6together with a pharmaceutically acceptable carrier therefor.
 10. Acomposition comprising(a) the composition represented by Formula III##STR20## (b) at least about two equivalents of a base (per mole of thecompound of Formula III) capable of forming a pharmaceuticallyacceptable salt with the compound of Formula III; (c) an amount ofhydroxypropyl-α, -β- or γ-cyclodextrin having about 2 to about 15hydroxypropyl groups per mole of said α-, β- and γ-cyclodextrin andwherein said amount is sufficient to achieve efficacious delivery ofsaid lipophilic oligosaccharide antibiotic to the serum of an animalwhile simultaneously avoiding occurrence of adverse reaction syndrome;and (d)0 to 6.0% by weight (basis a compound of Formula III) of apharmaceutically acceptable non-ionic surfactant.
 11. A pharmaceuticalcomposition for treating susceptible gram-positive and/or gram-negativebacterial infections comprising an antiinfective amount of a compositionof claim 10 and a pharmaceutically acceptable carrier therefor.
 12. Amethod of treating susceptible gram-positive and/or gram-negativebacterial infections in animals which comprises administering an amountof the pharmaceutical composition of claim 11 effective for suchpurpose.
 13. The pharmaceutical compositions of claim 11 wherein themolar ratio of (a):(b):(c) is 1:2-3:1-6.
 14. A method of preventingadverse reaction syndrome in animals following parenteral administrationof a lipophilic oligosaccharide antibiotic represented by Formula IIIwhile simultaneously delivering an anti-infective amount of a saidantibiotic to said animal, said method which comprises parenterallyadministering to said animal an anti-infective amount of a compositionof claim 10 together with a pharmaceutically acceptable carriertherefor.
 15. The composition of claim 10 wherein the base is selectedfrom the group consisting of chloroprocaine, procaine, piperazine,glucamine, N-methylglucamine, N'N-dimethylglucamine, ethylenediamine,diethanolamine, diisopropylamine, diethylamine,N-benzyl-2-phenylethylamine, N,N'-dibenzylethylenediamine, choline,clemizole, tris(hydroxymethyl)aminomethane, D-glucosamine and sodiumhydroxide.
 16. The composition of claim 10 wherein the base isN-methylglucamine.
 17. The composition of claim 10 whereinhydroxypropyl-β-cyclodextrin is used.
 18. A composition of claim 10which comprises a pharmaceutically acceptable non-ionic surfactant. 19.A composition of claim 18 wherein the pharmaceutically acceptablenon-ionic surfactant is a sorbitan mono-9-octa-decenoatepoly(oxy-1,2-ethanediyl) derivative.
 20. A composition of claim 10 whichfurther comprises manitol.
 21. A composition which comprises:(a) thecompound represented by Formula III: ##STR21## (b) N-methylglucamine;(c) hydroxyprophyl-β-cyctodextrin containing 7.4 hydroxypropyl groupsper molecule; (d) the sorbitan mono-9-octa-decenoatepoly(oxy-1,2-ethanediyl) derivate, polysorbate 80; and (e)manitol;wherein the molar ratio of (a):(b):(c) is 1:3:5and the weightpercent of the polysorbate 80 (basis the compound of formula III) isabout 2.85% of and the weight percent of manitol (basis wholecomposition) is about 18.28%.
 22. A method of treating susceptiblegram-positive and/or gram negative bacterial infections in animals whichcomprises administering an amount of the composition of claim 21effective for such treating.